Subproject #1 Selective Plane Illumination Microscopy for nematode neurodevelopment and minimally invasive in vivo imaging Selective plane illumination microscopy (SPIM (1)) is a technique whereby a sample is illuminated with a thin plane of light from the side, so that fluorescence detection occurs in a direction perpendicular to excitation. Such an experimental geometry has major advantages over conventional 3D microscopy techniques, such as confocal or 2 photon microscopy. First, acquisition speed is greatly increased relative to point-scanning methods, as the entire imaging plane is detected simultaneously. Second, excitation is confined to the focal plane, so each pixel is imaged only once during each volumetric acquisition. This drastically reduces light exposure and results in far lower photobleaching and photodamage than is possible with conventional imaging techniques. These advantages have been applied to studying whole-animal (zebrafish, drosophila) embryogenesis, and to the measurement of calcium transients in tissue slices. We have recently built a SPIM that we intend to use in constructing the first atlas of neuronal positions and migrations in the developing nematode C. Elegans, in collaboration with extramural researchers Daniel Colon-Ramos (Yale University), Zhirong Bao (Memorial Sloan-Kettering Cancer Center), and William Mohler (University of Connecticut). Due to the greatly reduced light dosage, we imaged nematode embryogenesis at 30x the speed of the best available competing technology (spinning disk confocal microscopy), with equivalent signal-to-noise ratio. This advance enabled the visualization of fast neurodevelopmental events in vivo (2). In collaboration with Kanta Subbarao (NIAID), we have also examined the 3D movement of GFP-tagged influenza particles in live cells, finding that viral RNA intermediates fuse in the cytoplasm before maturation into virions at the plasma membrane. We have now constructed a second generation SPIM instrument, where illumination and detection is conducted along two perpendicular views (dual-view inverted selective plane illumination microscopy, diSPIM). The advantage of the diSPIM is that axial resolution is significantly increased by merging the results obtained from each view. This setup enables isotropic imaging with 330 nm, more than quadrupling axial resolution compared to our previous instrument. We can maintain this high spatial resolution while also operating the microscope at high frame rates, up to 200 Hz in 2D and 2 Hz in 3D (for a 50 plane volume). The microscope enables high resolution, 4D imaging with minimal photobleaching and photodamage in cells and small embryos (3). We are in the process of writing a detailed methods paper that describes a DIY implementation that can be used by biologists to construct their own diSPIM. Subproject #2 Increasing the speed and depth penetration of structured illumination microscopy Structured illumination microscopy (SIM) is a super-resolution technique (4) that offers modest resolution improvement (2x better than the diffraction limit), but is readily compatible with live samples due to its low excitation intensities. SIM, while commercially available, is expensive and remains the province of relatively few labs. Furthermore, commercial SIM systems are limited to samples with thickness < 10 microns, as they do not physically reject background light. Together with Chris Combs (NHLBI), we developed a multifocal version of SIM (MSIM) that uses the confocal effect to image samples > 50 microns from the coverslip surface while maintaining resolution-doubling capability (5). MSIM enabled spatial resolutions down to 150 nm at frame rates of 1 Hz, at roughly 1/5 the cost of a commercial SIM. We have now developed an MSIM implementation that improves the speed of acquisition 100-fold, by performing most of the computational processing operations entirely in hardware (instant SIM, (6)). We also have actively collaborated with Dr. George Patterson (NIBIB) to implement a multiphoton version of MSIM that performs better in thicker samples (7), and we are working to develop a point-scanning version of the instant SIM that will enable even better depth penetration. (1) Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E. H. K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007-9 (2004). (2) Wu, Y. et al. Inverted selective plane illumination microscopy (iSPIM) enables coupled cell identity lineaging and neurodevelopmental imaging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 108, 17708-17713 (2011). (3) Wu, Y., Wawrzusin, P., Senseney, J., Fischer, R.S., Christensen, R., Santella, A., York, A.G., Winter, P.W., Waterman, C.M., Bao, Z., Colon-Ramos, D., McAuliffe, M., Shroff, H. Volumetric Isotropic Imaging with Dual-view Plane Illumination Microscopy. Nat. Biotechnol, in press. (4) Gustafsson, M.G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 198, 82-7 (2000). (5) York, A.G. et al. Resolution Doubling in Live, Multicellular Organisms via Multifocal Structured Illumination Microscopy. Nat. Methods 9, 749-754 (2012). See also code.google.com/p/msim/ (6) York, A.G., Chandris, P., Dalle Nogare, D., Head, J., Wawrzusin, P., Fischer, R.S., Chitnis, A., Shroff, H. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat. Methods, in press. (7) Ingaramo, M., York, A.G., Wawrzusin, P., Milberg, O., Hong, A., Weigert, R., Shroff, H., Patterson, G.H. Two-photon excitation improves resolution-doubled fluorescence microscopy in thick scattering tissue. PNAS, under revision.